Eukaryotic cells have evolved protective mechanisms that minimize the genotoxic effects of UVC-induced DNA lesions. These include nucleotide excision repair (NER), cell cycle checkpoints, and post-- replication repair (PRR). These systems operate in concert to promote genetic stability; deficiencies in any of them predispose humans to cancer. Xeroderma pigmentosum variant (XP-V) is a PRR mutant lacking DNA polymerase eta. This defect renders XP-V cells deficient in bypass replication of cyclobutane thymine dieters [(cis,syn)TT]. The capability of cell extracts (including XP-V) to bypass the other major UV photoproduct, the (6-4)TT, will be quantified. Duplex circular DNA molecules containing an undamaged di-thymine, a single (cis,syn)TT or a (6-4)TT, within the same sequence and on either side of the SV40 origin, will be constructed. A T:T mismatch nearby the dieter site will serve as a sequence marker for the complementary DNA strand. These substrates will be used to investigate the efficiency and mechanism of bypass replication of each photoproduct when it is encountered during leading or lagging strand synthesis. Delay or failure to bypass UVC- induced lesions leads to formation of daughter strand gaps. Blockage of DNA synthesis and/or formation of single strand DNA regions are postulated to signal to the S checkpoint, leading to inhibition of replicon initiation, extension of the S phase and possibly to S phase arrest. This project will establish whether ATM kinase, ATR kinase or both are involved in signal transduction pathways underlying S checkpoint responses to UVC. NER, PRR, and S checkpoint response are coordinated to promote completion of DNA replication and prevent formation of chromatid-type chromosomal aberrations. Although S phase XP-V cells are hypersensitive to UVC inhibition of DNA replication and to gene mutations, it less clear whether XP-V cells are abnormally sensitive to the clastogenic effects of UVC. UVC-induced chromosomal aberrations will be measured in normal and XP-V diploid fibroblasts immortalized by expression of human telomerase. The design and interpretation of these experiments will take into account the dose- and time-dependent effects of UVC on S phase cells. This project will provide insight on how PRR and the S checkpoint cooperate to protect human cells from cancer.